siRNA encoding constructs and methods for using the same

Abstract

siRNA encoding constructs and methods for using the same are provided. The subject constructs are characterized by including a siRNA coding domain flanked by opposing promoters. In using the subject constructs, sense and antisense strands of the desired siRNA product encoded by the coding domain are transcribed under the direction of the two opposing promoters flanking the coding domain. The transcribed sense and antisense strands are then annealed to each other to produce the desired siRNA double-stranded molecule. The subject constructs and methods find use in a variety of applications, including applications where the specific reduction or silencing of a gene is desired. Also provided are systems and kits for use in practicing the subject invention.

1 . A construct comprising two opposing promoters flanking a siRNA coding domain, wherein each of said promoters comprises a transcription terminator.  
     
     
         2 . The construct according to  claim 2 , wherein said transcription terminator is present in a non-transcribed region of each of said promoters.  
     
     
         3 . The construct according to  claim 2 , wherein in each of said promoters, said transcription terminator is adjacent to said siRNA coding domain.  
     
     
         4 . The construct according to  claim 3 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 20 nt.  
     
     
         5 . The construct according to  claim 4 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 10 nt.  
     
     
         6 . The construct according to  claim 5 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 5 nt.  
     
     
         7 . The construct according to  claim 6 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator ranges from about 0 to about 5 nt.  
     
     
         8 . The construct according to  claim 1 , wherein said siRNA coding domain comprises deoxyribonucleotides.  
     
     
         9 . The construct according to  claim 1 , wherein said coding domain encodes a siRNA double stranded molecule that is between about 20 and about 30 bp in length.  
     
     
         10 . The construct according to  claim 1 , wherein at least one of said two opposing promoters is an inducible promoter.  
     
     
         11 . The construct according to  claim 1 , wherein said construct is present on a vector.  
     
     
         12 . The construct according to  claim 11 , wherein said vector is a plasmid.  
     
     
         13 . The construct according to  claim 11 , wherein said vector is a viral vector.  
     
     
         14 . A method of producing a siRNA double-stranded molecule, said method comprising: 
 transcribing sense and anstisense RNA strands from a siRNA coding domain flanked by two opposing promoters so that said sense and antisense strands anneal to each other to produce said siRNA double-stranded molecule, wherein each of said promoters comprises a transcription terminator.    
     
     
         15 . The method according to  claim 14 , wherein said transcription terminator is present in a non-transcribed region of each of said promoters.  
     
     
         16 . The method according to  claim 15 , wherein in each of said promoters, said transcription terminator is adjacent to said siRNA coding domain.  
     
     
         17 . The method according to  claim 16 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 20 nt.  
     
     
         18 . The method according to  claim 17 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 10 nt.  
     
     
         19 . The method according to  claim 18 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 5 nt.  
     
     
         20 . The method according to  claim 19 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator ranges from about 0 to about 5 nt.  
     
     
         21 . The method according to  claim 14 , wherein said two opposing promoters flanking said coding sequence are present on a vector.  
     
     
         22 . The method according to  claim 21 , wherein said vector is a plasmid.  
     
     
         23 . The method according to  claim 21 , wherein said vector is a viral vector.  
     
     
         24 . The method according to  claim 14 , wherein said method is an in vitro method.  
     
     
         25 . The method according to  claim 14 , wherein said method is an in vivo method.  
     
     
         26 . The method according to  claim 14 , further comprising making said vector by: 
 (a) providing a provector comprising said two opposing promoters flanking at least one cloning site, and    (b) introducing said coding domain into said cloning site.    
     
     
         27 . The method according to  claim 26 , wherein said cloning site is a multiple cloning site.  
     
     
         28 . The method according to  claim 26 , wherein said cloning site comprises a sequence cleaved by a type IIS restriction endonuclease.  
     
     
         29 . The method according to  claim 14 , wherein at least one of said two opposing promoters is an inducible promoter.  
     
     
         30 . A method of making a construct that encodes a siRNA double-stranded molecule, said method comprising: 
 introducing a coding sequence for said siRNA double-stranded molecule into a cloning site of a proconstruct, wherein said cloning site is flanked by two opposing promoters, wherein each of said promoters comprises a transcription terminator.    
     
     
         31 . The method according to  claim 30 , wherein said transcription terminator is present in a non-transcribed region of each of said promoters.  
     
     
         32 . The method according to  claim 31 , wherein in each of said promoters, said transcription terminator is adjacent to said siRNA coding domain.  
     
     
         33 . The method according to  claim 32 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 20 nt.  
     
     
         34 . The method according to  claim 33 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 10 nt.  
     
     
         35 . The method according to  claim 34 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 5 nt.  
     
     
         36 . The method according to  claim 35 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator ranges from about 0 to about 5 nt.  
     
     
         37 . The method according to  claim 30 , wherein said cloning site is a multiple cloning site.  
     
     
         38 . The method according to  claim 30 , wherein said cloning site comprises at least one sequence cleaved by a type IIS restriction endonuclease.  
     
     
         39 . The method according to  claim 30 , wherein at least one of said two opposing promoters is an inducible promoter.  
     
     
         40 . A method of at least reducing the expression of a gene in a target cell, said method comprising: 
 introducing into said cell an effective amount of construct comprising two opposing promoters flanking a siRNA coding domain, wherein each of said promoters comprises a transcription terminator, under conditions sufficient to transcribe sense and anstisense RNA strands from said siRNA coding domain that anneal to each other to produce a siRNA double-stranded molecule for said gene that at least reduces expression of said gene.    
     
     
         41 . The method according to  claim 40 , wherein said transcription terminator is present in a non-transcribed region of each of said promoters.  
     
     
         42 . The method according to  claim 41 , wherein in each of said promoters, said transcription terminator is adjacent to said siRNA coding domain.  
     
     
         43 . The method according to  claim 42 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 20 nt.  
     
     
         44 . The method according to  claim 43 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 10 nt.  
     
     
         45 . The method according to  claim 44 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator is less than about 5 nt.  
     
     
         46 . The method according to  claim 45 , wherein the distance between the last nucleotide of said coding domain and the first nucleotide of said terminator ranges from about 0 to about 5 nt.  
     
     
         47 . The method according to  claim 40 , wherein said construct is present on a vector.  
     
     
         48 . The method according to  claim 47 , wherein said vector is a plasmid.  
     
     
         49 . The method according to  claim 47 , wherein said vector is a viral vector.  
     
     
         50 . The method according to  claim 40 , wherein said method is an in vitro method.  
     
     
         51 . The method according to  claim 40 , wherein said method is an in vivo method.  
     
     
         52 . The method according to  claim 40 , wherein said method is a method of silencing expression of said gene.  
     
     
         53 . The method according to  claim 40 , wherein said method is a loss of function assay.  
     
     
         54 . The method according to  claim 40 , wherein at least one of said two opposing promoters is an inducible promoter.  
     
     
         55 . A proconstruct comprising: 
 two opposing promoters flanking an insertion site, wherein each of said promoters comprises a transcription terminator.    
     
     
         56 . The proconstruct according to  claim 55 , wherein said transcription terminator is present in a non-transcribed region of each of said promoters.  
     
     
         57 . The proconstruct according to  claim 56 , wherein in each of said promoters, said transcription terminator is adjacent to said insertion site.  
     
     
         58 . The proconstruct according to  claim 56 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator is less than about 20 nt.  
     
     
         59 . The proconstruct according to  claim 58 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator is less than about 5 nt.  
     
     
         60 . The proconstruct according to  claim 60 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator ranges from about 0 to about 5 nt.  
     
     
         61 . The proconstruct according to  claim 55 , wherein said insertion site is a multiple cloning site.  
     
     
         62 . The proconstruct according to  claim 55 , wherein said insertion site comprises at least one sequence cleaved by a type IIS restriction endonuclease.  
     
     
         63 . The proconstruct according to  claim 55 , wherein at least one of said two opposing promoters is an inducible promoter.  
     
     
         64 . The proconstruct according to  claim 55 , wherein said proconstruct is a provector.  
     
     
         65 . The proconstruct according to  claim 64 , wherein said provector is a plasmid provector.  
     
     
         66 . The proconstruct according to  claim 64 , wherein said provector is a viral provector.  
     
     
         67 . A kit for use in preparing an RNAi double-stranded molecule, said kit comprising: 
 (a) a proconstruct comprising said two opposing promoters flanking an insertion site, wherein each of said promoters comprises a transcription terminator, and    (b) instructions for inserting a siRNA coding domain into said insertion site.    
     
     
         68 . The kit according to  claim 67 , wherein said transcription terminator is present in a non-transcribed region of each of said promoters.  
     
     
         69 . The kit according to  claim 68 , wherein in each of said promoters, said transcription terminator is adjacent to said insertion site.  
     
     
         70 . The kit according to  claim 69 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator is less than about 20 nt.  
     
     
         71 . The kit according to  claim 70 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator is less than about 10 nt.  
     
     
         72 . The kit according to  claim 71 , wherein the distance between the last nucleotide of said insertion stie and the first nucleotide of said terminator is less than about 5 nt.  
     
     
         73 . The kit according to  claim 72 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator ranges from about 0 to about 5 nt.  
     
     
         74 . The kit according to  claim 67 , wherein said kit further comprises a restriction endonuclease for said insertion site.  
     
     
         75 . The kit according to  claim 67 , wherein said insertion site is a multiple cloning site.  
     
     
         76 . The kit according to  claim 67 , wherein said insertion site comprises at least one sequence cleaved by a type IIS restriction endonuclease.  
     
     
         77 . The kit according to  claim 67 , wherein at least one of said two opposing promoters is an inducible promoter.  
     
     
         78 . A system for use in preparing an RNAi double-stranded molecule, said system comprising: 
 (a) a proconstruct comprising said two opposing promoters flanking an insertion site, wherein each of said promoters comprises a transcription terminator, and    (b) a restriction endonuclease for said cloning site.    
     
     
         79 . The system according to  claim 78 , wherein said transcription terminator is present in a non-transcribed region of each of said promoters.  
     
     
         80 . The system according to  claim 79 , wherein in each of said promoters, said transcription terminator is adjacent to said insertion site.  
     
     
         81 . The system according to  claim 80 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator is less than about 20 nt.  
     
     
         82 . The system according to  claim 81 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator is less than about 10 nt.  
     
     
         83 . The system according to  claim 82 , wherein the distance between the last nucleotide of said insertion stie and the first nucleotide of said terminator is less than about 5 nt.  
     
     
         84 . The system according to  claim 83 , wherein the distance between the last nucleotide of said insertion site and the first nucleotide of said terminator ranges from about 0 to about 5 nt.  
     
     
         85 . The system according to  claim 78 , wherein said insertion site is a multiple cloning site.  
     
     
         86 . The system according to  claim 78 , wherein said insertion site comprises at least one sequence cleaved by a type IIS restriction endonuclease.  
     
     
         87 . The system according to  claim 78 , wherein at least one of said two opposing promoters is an inducible promoter.